Biological control of wild poinsettia and other weedy spurges with a fungal pathogen

ABSTRACT

The subject invention concerns a novel bioherbicide and its use to control major weeds found in many fields in the United States. Specifically, Alternaria euphorbiicola Simmons &amp; Engelhard, having the identifying characteristics of deposit NRRL 18056, in an agricultural composition, can be used to effectively control wild poinsettia and weedy spurges. Further, Alternaria euphorbiicola Simmons &amp; Engelhard, having the identifying characteristics of deposit NRRL 18056, in a mixture with Alternaria cassiae can be used to control wild poinsettia and weedy spurges and other undesired vegetation, such as sicklepod, showy crotalaria and coffee senna. Further, the bioherbicide of the invention can be mixed with a chemical herbicide to increase weed control.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The instant invention is a method for the control of undesirable plantsby use of plant pathogens.

2. Description of the Prior Art

The merits for using plant pathogens to control weeds in annual cropshave been discussed previously for two Colletotrichum spp. (Daniel, etal. U.S. Pat. No. 3,849,104 and Templeton, U.S. Pat. No. 3,999,973). Theanthracnose fungus Colletotrichum gloeosporioides has been used tocontrol the weed northern jointvetch, and another strain of this fungushas been used to control winged waterprimrose. Colletotrichum malvarumhas been used to control prickly sida. These three pathogens have beencombined to control all three target weeds at once. In other work thefungus Alternaria cassiae (U.S. Pat. No. 4,390,360) has been used tocontrol sicklepod, coffee senna, and showy crotalaria. Another fungusFusarium lateritium (U.S. Pat. No. 4,419,120) has been used to controlprickly sida, velvetleaf and spurred anoda. Included in this samepatent, the synergistic interaction between F. lateritium and Alternariamacrospora has been used for control of spurred anoda.

Cercospora rodmanii has been used to control waterhyacinth (U.S. Pat.No. 4,097,261) and Phytophthora palmavora has been commerciallydeveloped as a biological herbicide for stranglervine.

Wild poinsettia (Euphorbia heterophylla L.) is a major weed problem inportions of the southern United States, Brazil, Columbia, Peru, Nigeria,and in several other countries which have tropical or subtropicalclimates (Akobundu, I.O. [1982] Weed Science 30: 331-334; Bannon, J. S.,Baker, J. B. and Rogers, R. L. [1978] Weed Science 26: 221-225; Reed, C.F. [1977] U.S. Dept. Agric. Handbook No. 498). This annual speciesreduces yield through direct competition with crop plants and interfereswith harvesting. The plants produce a sticky latex sap that interfereswith harvesting and reduces seed quality of soybeans by increasingmoisture levels and trash accumulation (Harger, T. R. and Nester, P. R.[1980] Louisiana Agric. 23(3): 4-5; Langston, V. B. and Harger, T. R.[1983] Proc. South. Weed Sci. Soc. 36: 77).

Wild poinsettia or "painted leaf" is an annual herb that commonly hastwo leaf shapes; long, narrow leaves, and wider, lobed leaves. Both leafshapes can occur on the same plants. As plants mature, the foliagedevelops numerous dark spots (Bannon et al., supra).

Seed germination is greatly influenced by light and temperature. Seedsremain viable in soil for extended periods of time, and maximumgermination rates occur as the soil temperatures increase in late springand early summer (Bannon et al. and Harger et al., supra).

Wild poinsettia is difficult and expensive to control using conventionalweed control practices. Planting soybeans early in the growing seasonbefore wild poinsettia seedlings emerge has been shown to provide somecontrol. Chemical herbicides recommended for control of wild poinsettiainclude metribuzin, linuron, acifluorfen, and dinoseb. Multipleherbicide applications are often necessary for satisfactory control(Harger et al., supra).

Spotted spurge (Euphoriba maculata L.) is an annual species that isnative to the eastern two-thirds of the United States. The leaves areoblong or lance-falcate, 0.8-3.5 cm long, with edges that are slightlytoothed. A conspicuous reddish spot usually occurs at the base of eachleaf. Spotted spurge is a major weed problem in soybeans, cotton, andother agronomic crops. Crop yields are reduced through directcompetition. In addition, the milky sap present in the stems causesdifficulty in harvesting.

Effective chemical control of spotted spurge is both difficult andexpensive. Norflurazon is used in cotton, and in soybeans the herbicidesused include acifluorfen, metribuzin, and dinoseb.

There is a need to have an effective means for the biological control ofwild poinsettia and weedy spurges, for example, spotted spurge. Such ameans would, advantageously, eliminate, or at least reduce the use ofchemical agents to control these weeds. It is well known that theextensive use of chemicals to control weeds over the last twenty yearshas placed a heavy burden on the ecosystem, resulting in contaminationof groundwater, lakes, rivers, etc. The substitution of biologicalcontrol agents for many of these chemicals is recognized as a solutionto this chemical contamination problem.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns the use of a novel isolate of the fungusAlternaria euphorbiicola Simmons & Engelhard to control the noxiousweeds wild poinsettia and weedy spurges. Advantageously, this funguscontrols these weeds when present among desired field crops withoutdamaging the crops. For example, the novel A. euphorbiicola isolate ofthe invention can be used to control wild poinsettia found in a fieldplanted with soybeans. Also, this novel isolate can be used to controlspotted spurge present in fields of soybeans, cotton, and otheragronomic crops. Though the prime use of the novel A. euphorbiicolaisolate is to control wild poinsettia and weedy spurges in fieldsplanted with desired crops, this fungus also can be used to controlthese weeds along roadways, river banks and lake shores, in recreationalareas, and the like.

DETAILED DESCRIPTION OF THE INVENTION

The novel A. euphorbiicola isolate of the subject invention is the firstknown biological control agent for controlling wild poinsettia and weedyspurges. This novel A. euphorbiicola isolate, as a biologically pureculture, is on deposit with the Agricultural Research Culture Collection(NRRL) in Peoria, Ill., and has been assigned the accession number NRRL18056. It was deposited on Mar. 12, 1986 to be maintained for 30 years.The address of the Curator of the Agricultural Research CultureCollection is: A. J. Lyons, Curator, ARS Patent Collection, NorthernRegional Research Center, 1815 North University Street, Peoria, Ill.61604.

According to E. G. Simmons, the description of the species is asfollows:

"Conidia from 5-day-old colonies on PCA initially are long-ovid,enlarging to long-ellipsoid and producing as many as six transversesepta in a 60-70×18 μm spore-body before a longitudinal septum is formedin one of the central cells. Only one (rarely two) longitudinal oroblique septum is produced in 1-4 of the central transverse compartmentsof a conidium. Conidia lack a definable beak as an entity distinct fromthe spore-body. In culture, as in the type, the apical conidium cellreadily lengthens and becomes converted into a functional conidiophore,a pseudorostrum; chains of as many as 4-5 or more units typically may beproduced through this mechanism. Conidia appear to be smooth, pale tan,with well-defined transverse septa and poorly defined longitudinal onesof weak appearance." (Simmons, E. G. [1986] Mycotaxon 25(1): 195-202)

Following are examples which illustrate the process of the invention,including the best mode. These examples should not be construed aslimiting. All solvent mixture proportions are by volume unless otherwisenoted.

EXAMPLE 1 Inoculum Production

Inoculum of A. euphorbiicola, NRRL 18056, for tests was produced inpetri dishes containing vegetable juice agar (V-8 juice, Campbell SoupCompany) in accordance with the method of P. M. Miller disclosed inPhytopathology 45: 461-2 (1955) in an article entitled "V-8 juice agaras a general-purpose medium for fungi and bacteria." The cultures wereincubated at 25° C. with a 12 hr diurnal light cycle supplied by two,40-w cool white fluorescent bulbs that were suspended 20 cm above thecultures. The 12 hr dark cycle temperature was 19° C. To produce largequantities of inoculum, conidia from petri-dish-grown cultures were usedto inoculate 500 ml of sterile liquid growth medium contained incotton-plugged 1000 ml Erlenmeyer flasks. The liquid growth mediumconsisted of soyflour, 15 g/L; corn meal, 15 g/L; sucrose, 30 g/L;calcium carbonate, 3 g/L and distilled water.

The cultures were incubated at 25° C. on a rotary shaker at 160 rpm.After 4 to 5 days, the mycelial cultures were harvested and homogenizedin a Waring Blendor for 30 sec. The mycelial homogenate was poured to adepth of 2 to 4 mm into shallow trays, and exposed to light from 250-wsunlamps for 5-15 min every 12 hr for 72 hr. The spores were vacuumedfrom the surface of the mycelial mat and stored at 4° C. Thissporulation procedure has been described in H. L. Walker and J. A. Riley(1982) Weed Sci 30: 651-654.

Granular preparations containing mycelia and conidia were prepared usingthe sodium alginate process described by H. L. Walker and W. J. Connick,Jr. (1983) Weed Sci 31: 333-338.

Mycelial fragment preparations were prepared by growing the fungus inliquid growth medium consisting of soyflour, 45 g/L; corn meal, 30 g/L;soluble starch, 15 g/L; sucrose, 30 g/L; calcium carbonate, 3 g/L; anddistilled water. The cultures were grown in Erlenmeyer flasks at 25° C.and 160 rpm. The mycelium was harvested 8 days after inoculation, andhomogenized for 30 sec in a Waring Blendor. Kaolin clay (1% w/v) wasadded, and the mycelial-clay mixture was homogenized for 15 sec. Themixture was centrifuged 1460 xg for 10 min and the supernatant wasdecanted. The mycelium-clay pellet was placed on filter paper and driedfor 5 to 7 days at 4° C. The dried cake was processed through a Wileymill (Arthur H. Thomas Company, Philadelphia, PA) with a 20 mesh screenand the resulting preparation was stored at 4° C.

EXAMPLE 2 Host Range and Epidemiology

The plant species included in the greenhouse studies are listed inTable 1. Plants were grown in a commercial potting mix (Mix No. 2, BallSeed Company, West Chicago, IL) in peat strips that contained 12 plantseach. Temperatures ranged from 28° to 32° C. with 40 to 60% relativehumidity. The day length was approximately 12 hr.

Plants in the cotyledon to third leaf stage of growth were sprayed torun off with inoculum applied with an atomizer. Inoculation mixturescontained 0.02% (v/v) surfactant, nonoxynol (9 to 10 PEO)[a(p-nonylphenyl)-w-hydroxypoly(oxyethylene)] in distilled water and1×10⁵ spores/ml. Control plants were sprayed with water and 0.02%surfactant only. All plants were placed in dew chambers for 20 hr at 25°C. The plants then were moved to greenhouse benches and evaluated dailyfor 14 days. All tests were repeated on at least two dates, and 12plants were used for each treatment in each test.

The fungus was pathogenic and highly virulent to wild poinsettiaseedlings. Most seedlings in the cotyledon to fourth leaf stage ofgrowth were killed 2 to 7 days affer inoculation. The pathogen produceddark brown to black lesions 1-5 mm in diam on the leaves and stemswithin 2 days. The lesions enlarged with time on any remaining plantsand produced severe stem canker and defoliation within seven days.Several other weedy spurge species appeared to be as susceptible as wildpoinsettia to the pathogen. Other representative crop and weed specieswere resistant to the pathogen; however, phytotoxic damage wasoccasionally observed on inoculated leaves of several species (Table 1).Phytotoxic symptoms ranged from flecking to a marginal or interveinal"burn" of inoculated leaves. These symptoms appeared within 48 to 72 hrafter inoculation and did not increase in number or severity with time.Succulent tissues were most susceptible to damage. The phytotoxicity isattributed to the high concentrations of conidia contained in theinoculation mixtures. Phytotoxic injury was not observed in every testand this injury was never observed on the control plants.

Wild poinsettia plants in all stages of growth were infected by thefungus; however, plants in the fourth leaf growth stage and younger weremost severely damaged (Table 2).

The fungus infected plants within a dew period temperature range of 10°to 35° C. (Table 3). At 25° C., the fungus infected with dew periodsranging from 0 to 24 hr (Table 4), and inoculum levels of 10,000 to500,000 spores per ml (Table 5).

This foliar pathogen can be formulated and applied to the target weedsas a spray (wettable powder) or as granules that consist of the fungusand a carrier such as vermiculite, corn cob grits, or clay.Advantageously, preemergence or postemergence applications of granulescan be used. The granular formulation of a foliar pathogen for soilapplication for preemergence weed control is unexpected becausesoil-inhabiting organisms compete with the pathogen.

The preferred liquid carrier is water, and the spore concentrate isdispersed to make a concentration of from about 1×10⁴ to about 1×10⁶spores/ml.

Spores of A. euphorbiicola can be mixed with those of Alternaria cassiaeto enlarge the scope of control of undesirable vegetation. For example,this mixture can be used to control both wild poinsettia and sicklepod(Cassia obtusifolia), two troublesome weeds in the Southeast. Further,spores of A. euphorbiicola can be mixed with those of A. cassiae tocontrol wild poinsettia and coffee senna. The use of A. cassiae tocontrol sicklepod, showy crotalaria and coffee senna is disclosed inU.S. Pat. No. 4,390,360, which is incorporated herein by referencethereto. The culture, means of growing, and application to these weedsdisclosed in U.S. Pat. No. 4,390,360 can be used herein. Mixtures of A.euphorbiicola and A. cassiae, for example, A. cassiae NRRL 12533, can bemade by methods well known in the art, utilizing the disclosure of U.S.Pat. No. 4,390,360 and that contained herein.

Though spores are the preferred form of the fungi, the fungi also can beformulated as fragmented mycelia and applied as foliar sprays.

Spores or mycelial fragments of A. euphorbiicola can be combined withvarious chemical additives, particularly chemical herbicides, toincrease weed control. These additives would be expected to broaden thespectrum of activity so that additional species of weeds can becontrolled. Application rates of these chemicals would be expected to beless than or equal to the rates recommended for conventional use.

Examples of these chemicals include but are not limited to thefollowing:

    ______________________________________                                                                       Common                                         Trade Name.sup.1                                                                        Chemical Name        Name                                           ______________________________________                                        Alanap (B)                                                                              2-[(1-naphthalenylamino)carbonyl]                                                                  naptalam                                                 berzoic acid                                                        Basagran (B)                                                                            Sodium salt of (3-isopropyl-1                                                                      bentazon                                                 H--2,1,3-bentzothiadiazin-4                                                                        sodium salt                                              (3H)--one 2,2-dioxide)                                              Basta (B & G)                                                                           Ammonium-DL-homoalanin-4-yl                                                                        glufosinate                                              (methyl) phosphinate ammonium                                       Blazer (B & G)                                                                          Sodium 5-[2-chloro-4-trifluoro                                                                     acifluorfen                                              methyl)phenoxy]-2-nitrobenzoate                                                                    sodium salt                                    Butyrac 200 (B)                                                                         4-(2,4-Dichlorophenoxy)butyric                                                                     2,4-DB                                                   acid                                                                Cobra (B) 1-(carboethoxy)ethyl 5-[2-chloro-                                                                  lactofen                                                 4-(trifluoromethyl)phenoxy]-2-                                                nitrobenzoate                                                       DOWPON (G)                                                                              2,2'-dichloropropionic acid                                                                        dalapon                                        Fusilade (G)                                                                            Butyl(R--S)-2-[4-[[5-(trifluoro-                                                                   fluazifop                                                methyl)-2-pyridinyl]oxy]phenoxy]                                              propanoate                                                          Hoelon (G)                                                                              Methyl 2-[4-(2,4-dichlorophenoxy)                                                                  diclofop                                                 phenoxy]propanoate   methyl                                         Premerge 3                                                                              Dinoseb(2-sec-butyl-4,6-dinitro-                                                                   dinoseb                                        (B & G)   phenol) as the alkanolamine                                                   salts                                                               Roundup   Isopropylamine salt of N--                                                                         glyphosate                                     (B & G)   (phosphonomethyl)glycine                                            Scepter (B)                                                                             Ammonium salt of 2-[4,5-Dihydro-                                                                   imazaquin                                                4-methyl ethyl)-5-oxo-1H--                                                    imidazol-2-yl]-3-quinoline                                                    carboxylic acid                                                     Classic   2-(([(4-chloro-6-methox-                                                                           DPX-F6025                                                pyrimidine-2-yl)amino carbonyl]                                               amino sulfonyl))benzoic acid                                                  ethyl ester                                                         Dual 8E   2-chloro-N--(2-ethyl-6-methyl-                                                                     metolachlor                                              phenyl)-N--(2-methoxy-1-methyl-                                               ethyl)acetamide                                                     Poast     2-[1-(ethoxyimino)butyl]-5[2-                                                                      sethoxydim                                               (ethylthio)propyl]-3-hydroxy-                                                 2-cyclohexen-1 one                                                  Sencor    4-Amino-6-(1,1-dimethylethyl)-                                                                     metribuzin                                               3-(methylthio)-1,2,4,-triazin-                                                5(4H)--one                                                          Lorox,    3-(3,4-dichlorophenyl)-1-                                                                          linuron                                        Linurex   methoxy-1-methylurea                                                Karmex    3-(3,4-dichlorophenyl)-1,1-                                                                        diuron                                                   dimethylurea                                                        Surflan   3 5-Dinitro-N.sup.4 N.sup.4 --dipropyl-                                                            oryzalin                                                 sulfanilamide                                                       B-Nine    Daminozide butanedioic acid                                                                        Alar                                                     mono(2,2-dimethylhydrazide)                                         Dropp     N--phenyl-N'--1,2,3-thiadiazol-                                                                    thidiazuron                                              5 yl urea                                                           Embark    Diethanolamine salt of (N--[2,4-                                                                   mefluidide                                               dimethyl-5-[[(trifluoromethyl)-                                               sulfonyl]amino]phenyl]acetamide                                     Stik      1-Naphthaleneacetic acid                                                                           NAA                                            ______________________________________                                         .sup.1 The notation in parentheses indicates the activity of the herbicid     (B = broadleaf control, G = grass control, and B & G = broadleaf and gras     control.                                                                 

                  TABLE 1                                                         ______________________________________                                        Reaction of Various Plant Species to Alternaria euphorbiicola.sup.a                             Disease Symptom                                                                         Plants                                                                De-     with                                              Family              folia-  stem    Disease                                   Species             tion    lesions Rating.sup.b                              ______________________________________                                        Cruciferae                                                                    Turnip ( Brassica rapa)                                                                           0       0       R                                         `Purple Top`                                                                  Cucurbitaceae                                                                 Watermelon (Citrullus vulgaris)                                                                   0       0       R                                         `Charleston Grey`                                                             Euphorbiaceae                                                                 Poinsettia (Euphorbia pulcherrima)                                                                0       0       S                                         Christmas Poinsettia                                                          Poinsettia (Euphorbia heterophylla)                                                               <10%    >95%    S                                         Mexican Fire Plant                                                            Poinsettia (Euphorbia heterophylla)                                                               >85%    >95%    S                                         Wild Poinsettia                                                               Spurge (Euphorbia hyssopifolia)                                                                   >85%    >95%    S                                         Spurge (Euphorbia polychroma)                                                                     0       0       S                                         Spurge (Euphorbia supina)                                                                         >85%    >95%    S                                         Prostrate Spurge                                                              Leguminosae                                                                   Beggarweed (Desmodium tortuosum)                                                                  0       0       R+                                        Florida Beggarweed                                                            Lima bean (Phaseolus limensis)                                                                    0       0       R                                         `Jackson Wonder`                                                              Cowpea (Vigna sinensis)                                                                           0       0       R                                         `California Pinkeye`                                                          Sicklepod (Cassia obtusifolia)                                                                    0       0       R+                                        Soybean (Glycine max)                                                         `Forrest`           0       0       R                                         ` Hill`             0       0       R                                         Malvaceae                                                                     Cotton (Gossypium hirsutum)                                                   `DPL-61`            0       0       R+                                        `Stoneville 50`     0       0       R+                                        Okra (Abelmoschus esculentus)                                                                     0       0       R+                                        `Clemson Spineless`                                                           Prickly sida (Sida spinosa)                                                                       0       0       R+                                        Velvetleaf (Abutilon theophrasti)                                                                 0       0       R                                         Solanaceae                                                                    Tomato (Lycopersicon esculentum)                                              `Better Boy`        0       0       R                                         `Manalucie`         0       0       R                                         `Rutgers`           0       0       R                                         ______________________________________                                         .sup.a Plants of each species were sprayed with inoculum containing 1         × 10.sup.5 spores/ml. Data were collected 14 days after inoculation     .sup.b R = resistant and S = susceptible to the pathogen; + = phytotoxic      injury by the pathogen limited to flecking or small, nondamaging burning      of the leaves.                                                           

                  TABLE 2                                                         ______________________________________                                        Effect of Growth Stage on Control of Wild Poinsettia by                       Alternaria euphorbiicola.sup.a                                                Growth Stage   Plants Killed (%)                                              ______________________________________                                        Cotyledon      33                                                             1 Leaf         83                                                             2 Leaves       100                                                            3 Leaves       50                                                             4 Leaves       83                                                             5 Leaves       17                                                             ______________________________________                                         .sup.a Twelve plants at each growth stage were sprayed to wetness with a      suspension containing 1 × 10.sup.5 conidia/ml. Plants received a        20hr dew period at 25° C. Data were collected 14 days after            inoculation.                                                             

                  TABLE 3                                                         ______________________________________                                        Effect of Different Dew-Period Temperatures on the Control                    of Wild Poinsettia by Alternaria euphorbiicola                                Temperature (C.)                                                                             Plants Killed (%)                                              ______________________________________                                        10             67                                                             15             50                                                             20             83                                                             25             92                                                             30              8                                                             35             17                                                             ______________________________________                                         .sup.a Twelve plants in the first to secondleaf state were sprayed to         wetness with a suspension containing 1× 10.sup.5 conidia/ml;            dewperiods were 20 hr. Data were collected 14 days after inoculation.    

                  TABLE 4                                                         ______________________________________                                        Effect of Dew-Period Duration on the Control of Wild Poin-                    settia by Alternaria euphorbiicola.sup.a                                      Length of                                                                     Dew-Period (hr)                                                                              Plants Killed (%)                                              ______________________________________                                        0              17                                                             4              58                                                             8              58                                                             12             75                                                             16             83                                                             20             100                                                            24             92                                                             ______________________________________                                         .sup.a Twelve plants in the first to second leaf stage of growth were         sprayed to wetness with a suspension that contained 1 × 10.sup.5        conidia/ml, then placed in dew chambers at 25° C. Data were            collected 14 days after inoculation.                                     

                  TABLE 5                                                         ______________________________________                                        Effect of Inoculum Levels on the Control of Wild Poinsettia                   with Alternaria euphorbiicola.sup.a                                           Spore Concentration                                                           (No./ml, × 10.sup.4)                                                                     Plants Killed (%)                                            ______________________________________                                        0                0                                                            1                33                                                           5                75                                                           10               92                                                           50               100                                                          ______________________________________                                         .sup.a Twelve plants in the first to secondleaf stage were inoculated wit     each spore concentration. Dewperiods were 20 hr at 25° C. Data wer     collected 14 days after inoculation.                                     

What is claimed is:
 1. A composition for controlling wild poinsettia andweedy spurges comprising a herbicidally effective amount of novelisolate of the fungus Alternaria euphorbiicola, having the identifyingcharacteristics of deposit NRRL 18056, in association with an inertagricultural carrier.
 2. A composition, according to claim 1, whereinsaid Alternaria euphorbiicola, having the identifying characteristics ofdeposit NRRL 18056, is in the spore form at a spore concentration offrom about 1×10⁴ spores/ml of carrier to about 1×10⁶ spores/ml ofcarrier.
 3. A process for controlling wild poinsettia or weedy spurgeswhich comprises applying a herbicidally effective amount of the fungusAlternaria euphorbiicola, having the identifying characteristics ofdeposit NRRL 18056, onto said wild poinsettia or weedy spurges or untothe situs of said wild poinsettia or weedy spurges.
 4. A process,according to claim 3, wherein said weedy spurge is spotted spurge.
 5. Abiologically pure culture of a novel isolate of Alternariaeuphorbiicola, having the identifying characteristics of deposit NRRL18056, and having the novel properties of pathogenicity to wildpoinsettia and weedy spurges.
 6. A process for controlling wildpoinsettia which comprises applying a herbicidally effective amount ofthe fungus Alternaria euphorbiicola, having the identifyingcharacteristics of deposit NRRL 18056, onto said wild poinsettia or untothe situs of said wild poinsettia.